Recombinant antigen from the NS3 region of the hepatitis C virus

ABSTRACT

The present invention concerns a polypeptide which is composed of the amino acids 1207 10 to 1488 10 of a hepatitis C virus and of less than 20 foreign amino acids and the use of this polypeptide as an antigen in an immunological test

This application is a Divisional of Ser. No. 08/511,759 filed Aug. 7,1995, U.S. Pat. No. 6,096,319.

The invention concerns polypeptides from the non-structural protein 3(NS3) region of the hepatitis C virus (HCV), a nucleic acid coding forsuch polypeptides as well as the use of the polypeptide as an antigen inan immunological test procedure or as helicase protein.

The disease denoted non-A-non-B hepatitis is caused in many cases by thehepatitis C virus (HCV). HCV is a single-stranded encapsulated RNA viruswhose genome is composed of about 9 to 10,000 bases. This genome codesfor structural proteins (core and envelope proteins) as well as fornon-structural proteins. The non-structural protein 3 (NS3) region ofHCV contains a protease and a helicase. The protease activity islocalized in the amino terminal third of the NS3 region.

A partial nucleotide sequence of HCV is disclosed in the European PatentApplication EP-A-0 318 216. It claims the use of nucleic acid fragmentsor polypeptide sections from HCV for a diagnostic test and fortherapeutic treatment.

EP-A-0 450 931 incorporated by reference discloses the completenucleotide and amino acid sequence of HCV. In addition a combination ofsynthetic HCV antigens is described comprising a first HCV antigen fromthe C domain and at least one further HCV antigen from one of thenon-structural domains NS3, NS4 or NS5 and the envelope domain S. Apreferred antigen from the domain NS3 is an antigen denoted C33c whichcomprises the amino acids 1192 to 1457 encoded by the HCV genome shownin FIG. 1 of EP-A-0 450 931.

The international Patent Application WO92/11370 concerns the cloning andsequencing of various polypeptides from the genome of HCV and the use ofthese polypeptides without any foreign protein parts in test kits and asa vaccine. A clone NS-3 deposited in connection with this application atthe “Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH,Mascheroder Weg 1b, 38124 Braunschweig, BRD (DSM)” under the number 6847contains the genetic information for a polypeptide of 527 amino acidsfrom the NS3 region of HCV.

Mori et al., (Jpn. J. Cancer Res. 83 (1992), 264-268) describes thediagnostic detection of a HCV infection by determining viral antibodiesin blood using viral proteins as antigens. These HCV proteins areexpressed in E. coli as a fusion protein with β-galactosidase. A proteinfrom the NS3 region containing the amino acids 1295 to 1541 of the HCVgenome showed the highest sensitivity. However, a disadvantage of suchfusion proteins is that cross-reactions with the fused protein part canoccur which reduce the specificity of the test reaction.

A test for HCV in blood requires a high degree of specificity andsensitivity. Furthermore the antigen used for the test should be capableof expression in high yield and be stable. Previously known antigensfrom the HCV genome have disadvantages since they do not fulfil one orseveral of the above requirements.

Thus an object of the present invention is to provide a polypeptideencoded by the HCV genome in which these disadvantages of the state ofthe art are at least partially eliminated and which, especially incomparison with known antigens, has a higher specificity and sensitivityand can be expressed in higher yield and is stable.

This object is achieved by a polypeptide which is composed of aminoacids 1207±10 to 1488±10 of a hepatitis C virus and has less than 20,preferably less than 15 foreign amino acids. The polypeptide accordingto the invention preferably contains the amino acids 1207±5 to 1488±5.More preferably they contain amino acids particularly preferably 1207±2to 1488±2 and most preferably 1207 to 1488 of hepatitis C virus in whichthe numbering of the amino acid residues refers to FIG. 1 of EP-A-0 450931 incorporated by reference.

The polypeptide according to the invention can be derived from anarbitrary HCV isolate, for example from a HCV isolate with a nucleotidesequence as described in EP-A-0 450 931. However, the polypeptide ispreferably derived from the HCV isolate from which the clone NS3described in WO92/11370 is derived which was deposited at the “DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH” (DSM), MascheroderWeg 1b, 38124 Braunschweig, under the number DSM 6847. The polypeptideaccording to the invention is preferably obtained by recombinantexpression of the vector pUC-D26.

SEQ ID NO:1 shows the amino acid sequence of a polypeptide according tothe invention. Amino acids 1-13 are foreign amino acids. Amino acids14-295 originate from HCV. The polypeptide according to the inventionpreferably contains amino acids 14-295 of the amino acid sequence shownin SEQ ID NO:1 and 2 or an amino acid sequence which is at least 90%homologous thereto.

The present invention also concerns a polypeptide defined as above whichcontains at least one marker group. All known marker groups come intoconsideration as the marker group that can be detected in a test systemi.e. directly or indirectly detectable marker groups. In this connectiona directly detectable marker group is understood as a group whichproduces a directly detectable signal e.g. a radioactive group, anenzyme group, a luminescence group, a metal complex etc.. On the otherhand the marker group can also be an indirectly detectable group e.g. abiotin or hapten group that is detectable by reaction with a suitablebinding partner (streptavidin, avidin or anti-hapten antibody) which inturn carries a signal-generating group. The marker group can be coupledin known manner to the antigen for example via a bifunctional spacer.Such processes for coupling marker groups to peptide antigens are knownto a person skilled in the area of immunology and do not need to bedescribed in detail here.

The polypeptide according to the invention preferably contains 1 to 12and particularly preferably 3 to 7 marker groups.

In addition the invention concerns a polypeptide as defined above inwhich one or several of the sulfhydryl groups of cysteine residues arepresent in covalently modified form. Examples of suitable covalentmodifying groups are maleimidodioxaoctylamine (MADOO),N-methyl-maleinimide (NMM), iodoacetic acid and iodoacetamide. Thecovalent cysteine modification results in particularly high specificimmunological reactivity.

Directly or indirectly detectable marker groups are preferablycovalently coupled to the sulfhydryl groups of the polypeptide. Examplesof SH-reactive bifunctional linkers for coupling to sulfhydryl groupsare maleimidopropylamine (MP), maleimidoethylamine (MEA) andmaleimidodioxaoctylamine (MADOO).

In addition, it may be preferable to use a polypeptide according to theinvention in which one or several cysteine residues are replaced byother natural or artificial amino acids. Cysteine residues arepreferably replaced by structurally analogous α-amino acids e.g. serineor α-aminobutyric acid. Cysteine substitutions lead to particularly highstability.

The polypeptide according to the invention has surprising advantagesover already known polypeptides. Compared to the antigen C33 describedin EP-A-0 450 931 which contains the amino acid residues 1192 to 1457 ofthe HCV sequence, the polypeptides according to the invention exhibit asubstantially higher specificity which is manifested in a statisticallysignificant lower number of false positive results in negative sera.Compared to the antigen NS3 described in WO92/11370 which contains theregion of amino acids 1007 to 1534 of the HCV sequence, the polypeptideaccording to the invention has considerably higher stability under testconditions. Compared to a polypeptide which contains amino acids 1227 to1528 from the NS3 region of HCV, the polypeptide according to theinvention has the advantage of improved expression efficiency and ahigher sensitivity. Due to these advantages the polypeptides accordingto the invention are substantially superior to all previously known HCVantigens from the NS3 region.

In addition the invention concerns an isolated nucleic acid which codesfor a polypeptide according to the invention. A preferred example ofsuch a nucleic acid is a foreign DNA encoding the peptide of interest inthe vector pUC-D26.

SEQ ID NO:1 also shows the nucleotide sequence of a nucleic acidaccording to the invention which codes for the polypeptides of SEQ IDNO:1 and 2. Nucleotides 40-885 code for the region of the polypeptidederived from HCV. The nucleic acid according to the invention preferablycontains (a) nucleotides 40-885 of the nucleotide sequence shown in SEQID NO:1 or (b) a nucleotide sequence that corresponds to a sequence from(a) within the scope of the degeneracy of the genetic code.

The present invention also concerns a vector which contains at least onecopy of a nucleic acid according to the invention. The vector accordingto the invention is preferably a prokaryotic vector i.e. a vectorsuitable for propagation in a prokaryotic host cell. Examples of suchvectors are shown in Sambrook et al. (Molecular Cloning. A LaboratoryManual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989)), especiallyin chapters 1 to 4 and 17. The vector according to the invention ispreferably a circular plasmid. The nucleic acid according to theinvention is preferably present on the vector under the control of apromoter sequence which allows expression of the polypeptide accordingto the invention. A preferred example of a vector according to theinvention is pUC-D26.

Furthermore the invention concerns a cell which is transformed with atleast one copy of a nucleic acid according to the invention or with avector according to the invention. The cell is preferably a prokaryoticcell, more preferably a gram-negative prokaryotic cell and mostpreferably an E. coli cell.

The polypeptide according to the invention is preferably used as anantigen in an immunological test procedure. On the other hand thepolypeptide can, however, also be used as a helicase protein and, due toits excellent antigenic action, for the production of a vaccine againstHCV infection.

The present invention additionally concerns a method for theimmunological determination of an antibody directed towards a hepatitisC virus in a sample liquid which is characterized in that the sampleliquid is incubated with at least one polypeptide according to theinvention and the antibody is detected via binding to the polypeptide.This immunological method of determination can be carried out using anyknown test format e.g. in a homogeneous immunoassay with a singlereaction phase or in a heterogeneous immunoassay with more than onereaction phase. A heterogeneous test format is preferably used in whichthe presence of the antibody is detected in the presence of a reactivesolid phase.

One embodiment of this test format is the so-called double antigenbridge test concept. In such a method the sample liquid is incubatedwith at least two polypeptides P₁ and P₂ according to the invention inwhich polypeptide P₁ (a) is bound to a solid phase or (b) is present ina form capable of binding to a solid phase and polypeptide P₂ carries amarker group. The antibody in the sample liquid is detected bydetermining the label in the solid phase or/and in the liquid phase,preferably in the solid phase, by means of an immobilized complex i.e.one that is bound to the solid phase.

The test procedure preferably comprises mixing the sample liquid with apurified labelled antigen P₂ and with the purified solid phase antigenP₁ in order to obtain a labelled, immobilized complex consisting oflabelled antigen, antibody and solid phase-bound antigen. Compared toother test formats for detecting antibodies, the bridge test formatleads to an improvement of the sensitivity of the assay, i.e. additionalimmunoglobulin classes such as IgM are recognized, and of thespecificity, i.e. unspecific reactivities with anti-IgG conjugate arereduced.

The labelled antigen P₂ carries a directly or indirectly detectablemarker group as described above. The solid phase antigen P₁ can be foundfor example, directly to the solid phase via a bifunctional spacer.However, P₁ is preferably a conjugate present in the solid phase of apolypeptide according to the invention and of a reaction partner of aspecific binding system. The other reaction partner of the specificbinding system is present bound to the solid phase. Examples of suchspecific binding systems are biotin/avidin, biotin/streptavidin,biotin/antibiotin, hapten/antihapten, carbohydrate/lectin and antibodyor antibody fragment and antibody against this antibody or against theantibody fragment. The antigen P₁ is preferably in the form of a biotinconjugate.

In such a double antigen bridge test the polypeptide antigen accordingto the invention is preferably used in a soluble form in order to avoidincreases in the blank value and an unfavourable signal/noise ratio dueto aggregations of the antigen. For this purpose the antigen is alreadyeither expressed in a soluble form in a suitable expression system, orafter expression in soluble form, it is renatured in vitro in a knownmanner. Furthermore in order to avoid the formation of covalentlycross-linked molecular aggregates, the immunological test can be carriedout under mild reducing conditions (addition of mild reducing reagents,preferably of sulfhydryl reagents, preferably DTT (dithiothreitol) orDTE (dithioerythritol) in a concentration range of 1 mmol/l to 25mmol/l) or/and preferably an antigen with covalently modified sulfhydrylgroups or/and an antigen with at least partially substituted cysteineresidues is used.

The polypeptide antigen according to the invention is preferably used ina soluble form in such a double antigen bridge test in order to avoidincreases in the blank and an unfavourable signal/noise ratio due toaggregations of the antigen. A detailed description of the bridge testformat is given in EP-A-0 280 211. This patent application isincorporated by reference.

The polypeptide according to the invention can, however, also be used inother test formats. An example of this is an indirect immunoassay forrecognizing a specific immunoglobulin by binding to an immobilizedspecific antigen and indirect detection via a conjugate with a secondantibody. In this embodiment of the method according to the inventionthe sample liquid is incubated with a polypeptide P₁ which is (a) boundto a solid phase or (b) is present in a form capable of binding to asolid phase and with a further antibody directed towards P₁ whichcarries a marker group. The antibody to be determined is detectedindirectly by determination of the label in the solid phase and/or inthe liquid phase, preferably in the solid phase. In this method thesignal produced on the solid phase by an immobilized complex of labelledantibody and solid phase-bound antigen is indirectly proportional to theconcentration of the antibodies to be determined in the sample liquid.

The present invention also concerns a reagent for the immunologicaldetermination of an antibody directed towards hepatitis C virus whichcontains at least one polypeptide according to the invention. If thereagent is used in a double antigen bridge test, it preferably containsat least two polypeptides P₁ and P₂ wherein polypeptide P₁ is (a) boundto a solid phase or (b) is present in a form capable of binding to asolid phase and polypeptide P₂ carries a marker group. Binding of thepolypeptide P₁ to the solid phase can either be achieved by directbinding or by means of a specific binding pair, preferablystreptavidin/avidin and biotin. The polypeptide P₁ is particularlypreferably present in a biotinylated form.

If it is used in an indirect immunological test the reagent according tothe invention preferably contains a polypeptide P₁ which is (a) bound toa solid phase or (b) is present in a form capable of binding to a solidphase and an antibody directed against P₁ which carries a marker group.The production of antibodies directed towards the polypeptide accordingto the invention is carried out in a well-known manner by immunizingexperimental animals with the corresponding antigen and isolatingpolyclonal antisera from the experimental animal. Alternatively amonoclonal antibody against the antigen can be produced by the method ofKöhler and Milstein or a further development of this method. Antibodyfragments or antibody derivatives can also be used instead of a completeantibody.

A further area of application of the polypeptide according to theinvention is to produce vaccines. For this the polypeptides according tothe invention are preferably produced in a purified form and thenbrought into the form of injectable liquids which can either besolutions or suspensions of the polypeptides. The polypeptides can alsobe enclosed in liposomes. Further constituents of these vaccines mayinclude water, salt solutions, glucose or glycerol. In addition thevaccines may contain small amounts of auxiliary substances such asemulsifiers, buffer substances, and if necessary adjuvants whichincrease the immune response. The vaccines are usually administeredparenterally by injection, preferably subcutaneously or intramuscularly.

Yet a further object of the present invention is a method for theimmunological determination of an antibody directed towards a hepatitisC virus in a sample liquid in which the sample liquid is incubated withat least one polypeptide that contains sequence regions from thehepatitis C virus, in particular from the NS3 region of the hepatitis Cvirus, and the antibody is detected by binding to the polypeptide, whichis characterized in that one uses a polypeptide from a region whichcontains at least one cysteine residue and (a) the antibody isdetermined under reducing conditions, (b) one or several cysteineresidues are covalently modified or/and (c) one or several cysteineresidues are replaced by other amino acids.

In contrast to other antigenic regions from HCV the NS3 region containsa particularly large accumulation of cysteine residues. Although the NS3protein synthesized during the course of virus replication is stable,the use of NS3 antigen s under physiological buffer conditions, e.g. inimmunological test procedures, has proven to be extremely problematicsince the free sulfhydryl groups of the cysteine residues easily oxidizeunder these conditions . This leads to intramolecular as well asintermolecular cross-linking of the a ntigen which significantly reducesits immunological reactivity.

Surprisingly it was possible to significantly improve the immunologic alreactivity of these NS3 antigens by means of a test format in which oneor several NS3 antigens with modified cysteine residues or/and withsubstituted cysteine residues are used or mild reducing conditions arepresent for example by addition of a sulfhydryl reagent. In this way anearly recognition of seroconversion is achieved as well as a significantamplification of the measurement signal.

The present invention is described further by the following examples andsequence protocols.

SEQ ID NO: 1: shows the nucleotide sequence of a preferred polypeptideaccording to the invention,

SEQ ID NO: 2: shows the amino acid sequence of a preferred nucleic acidaccording to the invention,

SEQ ID NO: 3: shows the nucleotide sequence of primer (1)

SEQ ID NO: 4: shows the nucleotide sequence of primer (2)

SEQ ID NO: 5: shows the nucleotide sequence of primer (3)

SEQ ID NO: 6: shows the nucleotide sequence of primer (4)

SEQ ID NO: 7: shows the nucleotide sequence of primer (5) and

SEQ ID NO: 8: shows the nucleotide sequence of primer (6).

EXAMPLES Example 1

Cloning and expression of a polypeptide with amino acids 1207 to 1488from the NS3 region of the hepatitis C virus

A DNA fragment was amplified by means of PCR starting with the clone NS3(DSM 6847) using primers (1) and (2) whose nucleotide sequence is shownin SEQ ID NO: 3 and SEQ ID NO: 4. Sequences for cloning (BamHI, BspHI,EcoRI restriction cleavage sites) are located at the 5′ end of this DNAfragment as well as an ATG codon and an AAA(Lys) codon to increaseexpression. Restriction sites for HindIII and EcoRI and a stop codon(TAA) are present at the 3′ end. In primers (1) and (2) the regionhomologous to HCV starts at nucleotide No. 19.

The DNA fragment obtained in this way was used in a pUC8 vector cleavedwith BamHI and HindIII. The resulting plasmid was denoted pUC-D26.

An E. coli strain JM109 (Yanisch-Perron et al., Gene 33 (1985), 103)transformed with plasmid pUC-D26 was incubated overnight in 100 mlmedium (L broth/ampicillin). Next morning the culture was diluted in a 3l flask with 900 ml 2× L-broth (10 g tryptone, 10 g yeast extract, 5 gNaCl per liter)/ampicillin. After addition of 2 ml glycerol and 1 to 2drops silicon anti-foam emulsion (Serva Company) the culture was thenshaken at ca. 185 rpm and 37° C. for 2 hours. Induction and antigenproduction were achieved by adding 2 mmol/l of the inducerisopropylthio-β-D-galactoside (IPTG) and shaking further for 3 to 4hours. Subsequently the bacteria were pelleted by centrifugation andprocessed further.

The bacterial pellets from two one liter cultures were resuspended in200 ml 50 mmol/l Tris-HCl, pH 8.5, 0.2 mg/ml lysozyme and 2 mmol/ldithioerythritol (DTE). Subsequently EDTA (final concentration: 15mmol/l), phenylmethylsulfonyl fluoride (final concentration: 1 mmol/l)and 4 mg DNase were added. The suspension was mixed for several minuteswith a magnetic stirrer and incubated for 45 minutes at 37° C. in awater bath.

Afterwards Triton-X100 (final concentration: 1%) was added and it wasstirred for 30 minutes in a magnetic stirrer. After freezing at −20° C.overnight and thawing, the cells were stirred for at least 1 hour at 37°C. and sonified if necessary. The incubation at 37° C. and/or ultrasonictreatment should be continued until the viscosity of the suspension oflysed cells has decreased significantly.

Subsequently this was centrifuged for 20 minutes at 35,000 g and 4° C.The resulting pellet was resuspended in 30 ml 50 mmol/l Tris-HCl, pH8.5, 2 mmol/l DTE, 150 mmol/l EDTA and 1.5% OGP(oxtyl-β-D-glucopyranoside, Biomol Company). This suspension was stirredvigorously at room temperature with a magnetic stirrer for at leastthree hours and subsequently centrifuged at 35,000 g and 4° C. for 20minutes.

The pellet was dissolved in 100 ml of 8 mol/l urea, 20 mmol/l Tris-HCl,pH 8.5, 2 mmol/l DTE and stirred. The antigen which was now dissolvedcan be frozen at −20° C. until further processing.

The protein was purified by the chromatographic steps described infrawhich were carried out at room temperature. The antigen was storedbetween each of the chromatographic steps at −20° C.

The first chromatographic step was carried out on a Q-Sepharose FastFlow column (Pharmacia) with a 20 mmol/l Tris-HCl pH 8.5, 8 mol/l urea,2 mmol/l DTE buffer. It was eluted with a NaCl gradient (0 to 0.7mol/l). The void volume and the fractions were tested by means of SDSPAGE. The polypeptide according to the invention is located in the firstmain peak. The positive fractions were pooled and dialyzed overnightagainst a 10-fold volume of 4 mol/l urea, 2 mmol/l DTE, 20 mmol/lTris-HCl pH 7.3.

The same column was used in the second chromatographic step. The columnbuffer was the dialysis buffer used after the first chromatographicstep. It was eluted using a NaCl gradient (0 to 0.5 mol/l) andsubsequently with 1 mol/l NaCl, NaOH pH 13, 4 mol/l urea.

The positive fractions were pooled and dialyzed overnight against thesame buffer as above. Finally it was chromatographed on a S-SepharoseFast Flow column (Pharmacia). The buffer and elution conditions were thesame as in the previous chromatographic step.

The antigen has a size of ca. 41 kDa on SDS polyacrylamide gel. Theyield was ca. 10 mg antigen per litre culture medium.

Example 2

Expression of the antigen according to the invention in comparison withother antigens from the HCV NS3 region

The following antigens were expressed:

a) antigen D26 according to the invention (amino acids 1207 to 1488)

b) antigen C33 (amino acids 1192 to 1457 according to EP-A 0 450 931)

c) antigen D27 (amino acids 1227 to 1528)

d) antigen NS3 (amino acids 1007-1534)

The antigen NS3 was expressed using the deposited clone NS-3 (DSM 6847).Cloning and expression of the antigens C33 and D27 were carried outaccording to the method described in example 1. The coding regions ofamino acids 1192 to 1457 for C33 and amino acids 1227 to 1528 for D27were amplified by standard methods using PCR starting with the plasmidpUC-N3 from the clone NS-3.

The primers (3) and (4) were used for C33 whose nucleotide sequences areshown in SEQ ID NO:5 and SEQ ID NO: 6. The primers (5) and (6) were usedfor D27 whose nucleotide sequences are shown in SEQ ID NO:7 and SEQ IDNO: 8. The region homologous to HCV starts at nucleotide No: 19 inprimers (3) and (5) and at nucleotide No. 13 in primers (4) and (6).

After treatment with the restriction enzymes BamHI and HindIII andsubsequent purification by agarose gel electrophoresis the amplified DNAfragments were inserted into the vector pUC8 that had been cleaved withBamHI and HindIII. Both antigens expressed by the pUC vectors have aregion of 13 non HCV-coded foreign amino acids at the N-terminal end(Met-Thr-Met-Ile-Thr-Asn-Ser-Arg-Gly-Ser-Ile-Met-Lys).

Afterwards the plasmids were transformed in E. coli JM109. (amino acids1-3 of SEQ ID NO:2) Clones which receive a DNA fragment coding for theC33 antigen express an antigen with a molecular weight of ca. 34 kDa(SDS-PAGE). In clones with a DNA fragment that codes for antigen D27, aband of 48 kDa is found.

In the case of C33 its percentage of the total protein is ca. 10% and isabout the same magnitude as the polypeptide D26 according to theinvention. A considerably smaller expression in the range of 5% or lessis found for D27.

Lysates of clones expressing D26, D27 and C33 were concurrentlyseparated in an SDS gel and transferred onto nitrocellulose. Afterincubating overnight with various HCV-positive sera in a 1:100 dilution,bound antibodies were detected by means of an anti-human IgGantibody-peroxidase conjugate and a colour reaction with3,3′-diaminobenzidine tetrachloride (DAB)/hydrogen peroxide.

Good reactivity is found in all HCV antigen segments with stronglypositive sera. In the case of weakly positive NS3-HCV sera the C33antigen exhibits in some cases the same and in other cases a slightlyweaker reactivity in comparison with the antigen according to theinvention. However, in the case of D27 a considerably reduced colourreaction with weak NS3-positive sera is found.

Example 3

Investigation of the specificity and sensitivity of various antigensfrom the NS3 region of HCV

The reactivity of various antigens from the NS3 region was comparativelyassessed on a total of 960 negative sera using the indirect testconcept. In this evaluation the antigen D26 according to the inventionwas found to have a significantly superior specificity compared to thereference antigen C33 which manifests itself as a significantly reducednumber of false positive results.

false positive correct negative Construct classification classificationC33 8 952 D26 2 958

In addition the reactivity of various NS3 constructs was comparativelyassessed using the indirect test concept on 20 sera of proven HCVstatus. A reduced sensitivity of antigen D27 was found with comparablesensitivity of

correct positive false negative Construct classification classificationNS3 20 0 C33 20 0 D26 20 0 D27 19 1

Example 4

Investigation of the stability of antigens from the NS3 region of HCV.

The stability of the antigens NS3 and D26 was comparatively evaluatedusing the indirect test concept. Antigen NS3 was found to have asignificantly poorer stability compared to antigen D26 according to theinvention. The stability of the antigens was investigated after 72 hoursincubation at 37° C.

Sample Construct No. Signal recovery NS3 1 <20% 2 <20% D26 1 99% 2 103%

Example 5

Investigation of the reactivity of the HCV-NS3 helicase antigen in thepresence or absence of reducing reagents

The time of seroconversion was tested on the bases of the reactivity ofserum samples collected at various times with the HCV-NS3 helicaseantigen under physiological conditions with or without 20 mmol/l DTT.The test concept was a double-antigen bridge test for theclass-independent recognition of all immunoglobulins in which an antigenprovided with an electrochemical marker group (ruthenium metal complex)and an antigen bindable to a solid phase (biotinylated antigen) wasused.

The results of this test are shown in the following table. It can beseen that it was possible to recognize seroconversion 38 days earlier inthe presence of 20 mmol/l DTT. In addition an improved signal strengthwas found in the presence of DTT.

TABLE Days after Reactivity of Reactivity of Day of start of the NS3antigen the NS3 antigen blood blood without DTT with DTT collectioncollection (signal/cut off) (signal/cut off) 28.07.1988 0 0.1 0.101.08.1988 4 0.1 0.1 08.08.1988 11 0.1 0.1 11.08.1988 14 0.1 0.115.08.1988 18 0.1 0.1 25.08.1988 28 0.1 0.1 29.08.1988 32 0.1 1.6*14.09.1988 48 0.1 6.5* 05.10.1988 69 1.3* 4.8* 19.10.1988 83 2.1* 6.8**positive signal

9 885 base pairs nucleic acid both linear cDNA hepatitis C virus NS3 CDS1..885 1 ATG ACC ATG ATT ACG AAT TCC CGG GGA TCC ATC ATG AAA TCC CCG GTG48 Met Thr Met Ile Thr Asn Ser Arg Gly Ser Ile Met Lys Ser Pro Val 1 510 15 TTC ACG GAT AAC TCC TCT CCA CCG GTA GTG CCC CAG AGC TTC CAG GTG 96Phe Thr Asp Asn Ser Ser Pro Pro Val Val Pro Gln Ser Phe Gln Val 20 25 30GCT CAC CTG CAT GCT CCC ACA GGC AGC GGC AAG AGC ACC AAG GTC CCG 144 AlaHis Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro 35 40 45 GCTGCA TAC GCA GCT CAG GGC TAC AAG GTG CTA GTG CTC AAC CCT TCT 192 Ala AlaTyr Ala Ala Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser 50 55 60 GTT GCTGCA ACA TTG GGC TTT GGT GCC TAC ATG TCC AAG GCT CAT GGG 240 Val Ala AlaThr Leu Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly 65 70 75 80 ATC GATCCT AAC ATC AGG ACC GGG GTG AGA ACA ATT ACC ACT GGC AGC 288 Ile Asp ProAsn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser 85 90 95 CCC ATT ACGTAC TCC ACT TAC GGC AAG TTT CTT GCC GAC GGC GGG TGC 336 Pro Ile Thr TyrSer Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys 100 105 110 GCA GGG GGTGCT TAT GAC ATA ATA ATT TGT GAC GAG TGC CAC TCC ACG 384 Ala Gly Gly AlaTyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr 115 120 125 GAT GCC ACATCC ATC TTG GGC ATC GGC ACT GTC CTT GAC CAA GGA GAG 432 Asp Ala Thr SerIle Leu Gly Ile Gly Thr Val Leu Asp Gln Gly Glu 130 135 140 ACT GCG GGGGCG AAA TTG GTT GTG TTC GCC ACC GCC ACC CCT CCG GGC 480 Thr Ala Gly AlaLys Leu Val Val Phe Ala Thr Ala Thr Pro Pro Gly 145 150 155 160 TCC GTCACT GTG CCC CAT CCC AAC ATT GAG GAG GTT GCT CTA TCC ACC 528 Ser Val ThrVal Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Thr 165 170 175 ACC GGAGAG ATC CCT TTT TAC GGC AAG GCT ATC CCC CTT GAG GTA ATC 576 Thr Gly GluIle Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu Val Ile 180 185 190 AAG GGGGGG AGA CAT CTC ATC TTC TGT CAT TCA AAG AGG AAG TGC GAT 624 Lys Gly GlyArg His Leu Ile Phe Cys His Ser Lys Arg Lys Cys Asp 195 200 205 GAG CTCGCC ACA AAG CTG GTC GCA ATG GGC ATC AAT GCC GTG GCC TAC 672 Glu Leu AlaThr Lys Leu Val Ala Met Gly Ile Asn Ala Val Ala Tyr 210 215 220 TAC CGCGGT CTT GAC GTG TCC GTC ATC CCG ACC AGC GGT GAT GTT GTC 720 Tyr Arg GlyLeu Asp Val Ser Val Ile Pro Thr Ser Gly Asp Val Val 225 230 235 240 GTCGTG GCA ACC GAC GCC CTC ATG ACC GGC TAT ACC GGC GAC TTC GAC 768 Val ValAla Thr Asp Ala Leu Met Thr Gly Tyr Thr Gly Asp Phe Asp 245 250 255 TCGGTG ATA GAC TGC AAC ACG TGT GTC ACT CAG ACA GTC GAT TTC AGC 816 Ser ValIle Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser 260 265 270 CTTGAC CCT ACC TTC ACC ATT GAG ACG ACC ACA CTT CCC CAG GAT GCT 864 Leu AspPro Thr Phe Thr Ile Glu Thr Thr Thr Leu Pro Gln Asp Ala 275 280 285 GTCTCC CGC ACT CAA CGA CGG 885 Val Ser Arg Thr Gln Arg Arg 290 295 295amino acids amino acid linear protein not provided 2 Met Thr Met Ile ThrAsn Ser Arg Gly Ser Ile Met Lys Ser Pro Val 1 5 10 15 Phe Thr Asp AsnSer Ser Pro Pro Val Val Pro Gln Ser Phe Gln Val 20 25 30 Ala His Leu HisAla Pro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro 35 40 45 Ala Ala Tyr AlaAla Gln Gly Tyr Lys Val Leu Val Leu Asn Pro Ser 50 55 60 Val Ala Ala ThrLeu Gly Phe Gly Ala Tyr Met Ser Lys Ala His Gly 65 70 75 80 Ile Asp ProAsn Ile Arg Thr Gly Val Arg Thr Ile Thr Thr Gly Ser 85 90 95 Pro Ile ThrTyr Ser Thr Tyr Gly Lys Phe Leu Ala Asp Gly Gly Cys 100 105 110 Ala GlyGly Ala Tyr Asp Ile Ile Ile Cys Asp Glu Cys His Ser Thr 115 120 125 AspAla Thr Ser Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Gly Glu 130 135 140Thr Ala Gly Ala Lys Leu Val Val Phe Ala Thr Ala Thr Pro Pro Gly 145 150155 160 Ser Val Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu Ser Thr165 170 175 Thr Gly Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro Leu Glu ValIle 180 185 190 Lys Gly Gly Arg His Leu Ile Phe Cys His Ser Lys Arg LysCys Asp 195 200 205 Glu Leu Ala Thr Lys Leu Val Ala Met Gly Ile Asn AlaVal Ala Tyr 210 215 220 Tyr Arg Gly Leu Asp Val Ser Val Ile Pro Thr SerGly Asp Val Val 225 230 235 240 Val Val Ala Thr Asp Ala Leu Met Thr GlyTyr Thr Gly Asp Phe Asp 245 250 255 Ser Val Ile Asp Cys Asn Thr Cys ValThr Gln Thr Val Asp Phe Ser 260 265 270 Leu Asp Pro Thr Phe Thr Ile GluThr Thr Thr Leu Pro Gln Asp Ala 275 280 285 Val Ser Arg Thr Gln Arg Arg290 295 40 base pairs nucleic acid single linear cDNA not provided 3AAGGGATCCA TCATGAAATC CCCGGTGTTC ACGGATAACT 40 39 base pairs nucleicacid single linear cDNA not provided 4 GGGAAGCCTT AATTCTTACC GTCGTTGAGTGCGGGAGAC 39 39 base pairs nucleic acid single linear cDNA not provided5 GAGGGATCCA TCATGAAAGC GGTGGACTTT ATCCCTGTG 39 33 base pairs nucleicacid single linear cDNA not provided 6 GAGAAGCTTT TAACACGTGT TGCAGTCTATCAC 33 39 base pairs nucleic acid single linear cDNA not provided 7GAGGGATCCA TCATGAAACA CCTGCATGCT CCCACCGGC 39 33 base pairs nucleic acidsingle linear cDNA not provided 8 GAGAAGCTTT TAATACCAAG CACAGCCTGC GTC33 302 amino acids amino acid linear protein not provided 9 Pro Val GluAsn Leu Glu Thr Thr Met Arg Ser Pro Val Phe Thr 1 5 10 15 Asp Asn SerSer Pro Pro Val Val Pro Gln Ser Phe Gln Val Ala 20 25 30 His Leu His AlaPro Thr Gly Ser Gly Lys Ser Thr Lys Val Pro 35 40 45 Ala Ala Tyr Ala AlaGln Gly Tyr Lys Val Leu Val Leu Asn Pro 50 55 60 Ser Val Ala Ala Thr LeuGly Phe Gly Ala Tyr Met Ser Lys Ala 65 70 75 His Gly Ile Asp Pro Asn IleArg Thr Gly Val Arg Thr Ile Thr 80 85 90 Thr Gly Ser Pro Ile Thr Tyr SerThr Tyr Gly Lys Phe Leu Ala 95 100 105 Asp Gly Gly Cys Ala Gly Gly AlaTyr Asp Ile Ile Ile Cys Asp 110 115 120 Glu Cys His Ser Thr Asp Ala ThrSer Ile Leu Gly Ile Gly Thr 125 130 135 Val Leu Asp Gln Gly Glu Thr AlaGly Ala Lys Leu Val Val Phe 140 145 150 Ala Thr Ala Thr Pro Pro Gly SerVal Thr Val Pro His Pro Asn 155 160 165 Ile Glu Glu Val Ala Leu Ser ThrThr Gly Glu Ile Pro Phe Tyr 170 175 180 Gly Lys Ala Ile Pro Leu Glu ValIle Lys Gly Gly Arg His Leu 185 190 195 Ile Phe Cys His Ser Lys Arg LysCys Asp Glu Leu Ala Thr Lys 200 205 210 Leu Val Ala Met Gly Ile Asn AlaVal Ala Tyr Tyr Arg Gly Leu 215 220 225 Asp Val Ser Val Ile Pro Thr SerGly Asp Val Val Val Val Ala 230 235 240 Thr Asp Ala Leu Met Thr Gly TyrThr Gly Asp Phe Asp Ser Val 245 250 255 Ile Asp Cys Asn Thr Cys Val ThrGln Thr Val Asp Phe Ser Leu 260 265 270 Asp Pro Thr Phe Thr Ile Glu ThrThr Thr Leu Pro Gln Asp Ala 275 280 285 Val Ser Arg Thr Gln Arg Arg GlyArg Thr Gly Arg Gly Lys Pro 290 295 300 Gly Ile 302

What is claimed is:
 1. Method for determining a hepatitis C virusspecific antibody in a sample, comprising: incubating said sample withat least one peptide derived from a hepatitis C virus protein NS3 regionwherein said at least one peptide has higher immunological activity withsaid hepatitis C virus specific antibody than a polypeptide consistingof an amino acid sequence from an NS3 protein region of a naturallyoccurring hepatitis C virus, wherein said at least one peptide has atleast one covalently modified cysteine residue or at least one cysteineresidue has been replaced by a different amino acid to form a peptidehaving a sequence not found in any HCV isolate, and determining bindingof said antibody to said at least one peptide.
 2. The method of claim 1,wherein said cysteine residue has been modified via covalent attachmentof a modifying group.
 3. The method of claim 1, wherein said cysteineresidue is modified by replacing it with another natural or artificialamino acid.
 4. The method of claim 3, wherein said cysteine residue hasbeen replaced by serine or α-aminobutyric acid.
 5. The method of claim1, wherein said at least one cysteine residue is modified by a modifyinggroup selected from the group consisting of maleimidodioctylamine,N-methyl-maleinimide, iodoacetic acid, and iodoacetamide, wherein saidmodifying group is covalently bound to said at least one cysteineresidue.
 6. The method of claim 1, wherein said polypeptide consists ofat least amino acids 19 to 290 of SEQ ID NO: 9, and no more than aminoacids 9 to 300 of SEQ ID NO:
 9. 7. The method of claim 1, wherein saidpolypeptide consists of at least amino acids 16 to 293 of SEQ ID NO: 9and no more than amino acids 12 to 297 of SEQ ID NO:
 9. 8. The method ofclaim 1, wherein said polypeptide consists of amino acids 14 to 295 ofSEQ ID NO:
 2. 9. The method of claim 1, further comprising incubatingsaid sample under reducing conditions which prevent formation ofcovalent, cross linked molecular aggregates.
 10. The method of claim 1,wherein said peptide consists of at least amino acids 21-282 and no morethan amino acids 1-302 of SEQ ID NO:
 9. 11. A method for determining ahepatitis C virus specific antibody in a sample, comprising incubatingsaid sample, under reducing conditions with an isolated polypeptidewhich consists of (a) at least amino acids 21-282 of SEQ ID NO: 9 and nomore than amino acids 1-302 of SEQ ID NO: 9 and (b) a contiguoussequence of less than 20 amino acids which is not found in hepatitis Cvirus proteins, wherein (b) is concatenated to the N or C terminus of(a), or an isolated polypeptide which is at least 90% identical to SEQID NO: 9, wherein at least one cysteine of said amino acids is modified,either by replacing it with another natural or artificial amino acid, orbv a modifying group and determining any binding to said polypeptide asa determination of said antibody.
 12. The method of claim 11, whereinsaid at least one cysteine is modified by a modifying group selectedfrom the group consisting of maleimidodioctylamine, N-methylmaleinimide, iodoacetic acid, and iodoacetamide, wherein said modifyinggroup is covalently bound to said at least one cysteine residue.
 13. Themethod of claim 11, wherein said at least on cysteine has been replacedby serine or α-aminobutyric acid.
 14. Method for determining a hepatitisC virus specific antibody in a sample comprising incubating said sample,under reducing conditions which prevent formulation of covalent, crosslinked molecular aggregates, with at least one polypeptide derived froma hepatitis C virus NS3 region, consisting of at least amino acids21-282 and no more than amino acids 1-302 of SEQ ID NO: 9 which isimmunologically reactive with said hepatitis C virus specific antibody,and determining binding of said antibody to said peptide.
 15. The methodof claim 14, wherein said polypeptide consists of amino acids 14-295 ofSEQ ID NO:
 2. 16. The method of claim 14, wherein said polypeptideconsists of at least amino acids 16-287 and no more than amino acids6-297 of SEQ ID NO:
 9. 17. The method of claim 14, wherein saidpolypeptide consist of at least amino acids 13-290 and no more thanamino acids 9-294 of SEQ ID NO: 9.